This invention relates generally to the field of recombinantly-produced proteins and specifically to novel, recombinant Entomopoxvirus genes, proteins, protein regulatory sequences and their uses in expressing heterologous genes in transformed hosts.
Poxviruses are taxonomically classified into the family Chordopoxvirinae, whose members infect vertebrate hosts, e.g., the Orthopoxvirus vaccinia, or into the family Entomopoxvirinae. Very little is known about members of the Entomopoxvirinae family other than the insect host range of individual members. One species of Entomopoxvirus (EPV) is the Amsacta moorei Entomopoxvirus (AmEPV), which was first isolated from larvae of the red hairy caterpillar Amsacta moorei (Roberts and Granados [1968] J. Invertebr. Pathol. 12:141-143). AmEPV is the type species of genus B of EPVs and is one of three known EPVs which will replicate in cultured insect cells (R. R. Granados et al. [1976] xe2x80x9cReplication of Amsacta moorei Entomopoxvirus and Autographa californica Nuclear Polyhedrosis Virus in Hemocyte Cell Lines from Estigmene acrea,xe2x80x9d in Invertebrate Tissue Culture Applications in Medicine, Biology, and Agriculture, E. Kurstak and K. Maramorosch (ed.), Academic Press, New York, pp. 379-389; T. Hukuhara et al. [1990] J. Invertebr. Pathol. 56:222-232; and Sato, T. [1989] xe2x80x9cEstablishment of Eight Cell Lines from Neonate Larvae of Torticids (Lepidoptera), and Their Several Characteristics Including Susceptibility to Insect Viruses,xe2x80x9d in Invertebrate Cell Systems Applications, J. Mitsuhashi (ed.), Vol. II, CRC Press, Inc., Boca Raton, Fla., pp. 187-198).
AmEPV is one of the few insect poxviruses which can replicate in insect cell culture; AmEPV is unable to replicate in vertebrate cell lines. The AmEPV double-stranded DNA genome is about 225 kb and is unusually A+T rich (18.5% G+C) (W. Langridge, H. R., et al. [1977] Virology 76:616-620). Recently, a series of restriction maps for AmEPV were published (Hall, R. L., et al. [1990] Arch. Virol. 110:77-90). No DNA homology to vaccinia has been detected (Langridge, W. H. [1983] J. Invertebr. Pathol. 42:77-82; Langridge, W. H. [1984] J. Invertebr. Pathol. 43:41-46).
The viral replication cycle of AmEPV resembles that of other poxviruses except for the appearance of occluded virus late in infection. For AmEPV, once a cell is infected, both occluded and extracellular virus particles are generated. The mature occlusion body particle, which is responsible for environmentally protecting the virion during infection, consists of virus embedded within a crystalline matrix consisting primarily of a single protein, spheroidin. Spheroidin, the major structural protein of AmEPV, has been reported to be 110 kDa in molecular weight and to consist of a high percentage of charged and sulfur-containing amino acids (Langridge and Roberts [1982] J. Invertebr. Pathol. 39:346-353).
Another insect virus is the baculovirus. Like baculoviruses, a characteristic feature of entomopoxviruses is the amalgamation of virions within environmentally stable occlusion bodies. It is this occluded form of the virus that is primarily responsible for dissemination to other insects. While the major protein (polyhedrin) of baculovirus occlusions is quite similar between viruses, it has been reported that the major occlusion body protein (spheroidin) of two group B entomopoxviruses, Amsacta moorei (AmEPV) and Choristoneura biennis (CbEPV) is quite different both in terms of amino acid sequence and coding capacity of the corresponding spheroidin genes (115 and 47 kDa for AmEPV and CbEPV, respectively).
The entomopoxviruses and the role of occlusion bodies have recently been reviewed by Arif and Kurstak (Arif, B. M., E. Kurstak, E. [1991 ] xe2x80x9cThe entomopoxviruses,xe2x80x9d In Viruses of Invertebrates (E. Kurstak, Ed.), pp. 179-195, Marcel Dekker, Inc., New York) and Goodwin et al. (Goodwin, R. H., R. J. Milner, C. D. Beaton [1991] xe2x80x9cEntomopoxvirinae,xe2x80x9d In Atlas of Invertebrate Viruses (J. R. Adams and J. R. Bonami, Eds.), pp. 259-285, CRC Press, Inc., Boca Raton). The gene which encodes the AmEPV spheroidin, a 115 kDa protein, has been identified and sequenced (Hall, R. L., R. W. Moyer [1991] J. Virol. 65, 6516-6527; Banville, M., F. Dumas, F., S. Trifiro, B. Arif, C. Richardson [1992] J. Gen. Virol. 73, 559-566). The AmEPV gene was also mapped and found to be located at the 3xe2x80x2 end of a nucleoside triphosphate phosphohydrolase gene (NPH I or NTPase I, Hall and Moyer [1991], supra). The spheroidin gene of Choristoneura biennis entomopoxvirus (CbEPV) has been reported to be derived from a gene capable of encoding a 47 kDa protein (Yuen, L., J. Dionne, B. Arif, C. Richardson [1990] Virology 175:427-433.). A comparison of the sequence of the two spheroidins shows no relationship between the two encoded proteins.
We have investigated the spheroidin genes of Choristoneura biennis, Choristoneura fumiferana, and Amsacta moorei viruses. Our results indicate, in contrast to published results, that the initial Choristoneura EPV spheroidin assignment is likely incorrect and that the Choristoneura spheroidin is instead a highly conserved homolog of the AmEPV spheroidin.
The use of viruses and virus proteins in eukaryotic host-vector systems has been the subject of a considerable amount of investigation and speculation. Many existing viral vector systems suffer from significant disadvantages and limitations which diminish their utility. For example, a number of eukaryotic viral vectors are either tumorigenic or oncogenic in mammalian systems, creating the potential for serious health and safety problems associated with resultant gene products and accidental infections. Further, in some eukaryotic host-viral vector systems, the gene product itself exhibits antiviral activity, thereby decreasing the yield of that protein.
In the case of simple viruses, the amount of exogenous DNA which can be packaged into a simple virus is limited. This limitation becomes a particularly acute problem when the genes used are eukaryotic. Because eukaryotic genes usually contain intervening sequences, they are too large to fit into simple viruses. Further, because they have many restriction sites, it is more difficult to insert exogenous DNA into complex viruses at specific locations.
Vaccinia virus has recently been developed as an eukaryotic cloning and expression vector (Mackett, M., et al. [1985] DNA Cloning, Vol. II, ed. D. M. Glover, Oxford: IRL Press, pp. 191-212; Panicali, D., et al. [1982] Proc. Natl. Acad. Sci. USA, 88:5364-5368). Numerous viral antigens have been expressed using vaccinia virus vectors (Paoletti, E., et al. [1984] Proc. Natl. Acad. Sci. USA 81:193-197; Piccine, A., et al. [1986] BioEssays 5:248-252) including, among others, HBsAg, rabies G protein and the gp120/gp41 of human immunodeficiency virus (HIV). Regulatory sequences from the spruce budworm EPV have been used previously with vaccinia (Yuen, L., et al. [1990] Virology 175:427-433).
Additionally, studies with vaccinia virus have demonstrated that poxviruses have several advantageous features as vaccine vectors. These include the ability of poxvirus-based vaccines to stimulate both cell-mediated and humoral immunity, minimal cost to mass produce vaccine and the stability of the lyophilized vaccine without refrigeration, ease of administration under non-sterile conditions, and the ability to insert at least 25,000 base pairs of foreign DNA into an infectious recombinant, thereby permitting the simultaneous expression of many antigens from one recombinant.
There exists a need in the art for additional viral compositions and methods for use in expressing heterologous genes in selected host cells, and in performing other research and production techniques associated therewith. In addition, it is noted that the host range of entomopoxviruses is restricted to specific insect hosts which differ from the host range of the baculovirus. Thus, for environmental control of certain pests provision of recombinant entomopoxviruses is desirable.
This invention pertains to novel vectors useful for producing proteins via the expression of an heterologous gene in a novel expression system. More particularly, this invention relates to methods for incorporating a selected heterologous gene (also referred to as exogenous DNA) into a poxvirus genome to produce a recombinant expression vector capable of expression of the selected gene in a host cell.
The expression systems described herein utilize novel structural and/or regulatory DNA elements from Entomopoxvirus genomes. For example, according to the subject invention, the entomopoxvirus spheroidin gene and/or the thymidine kinase gene can be used as the location for the insertion of exogenous DNA. These Entomopoxvirus genes have been discovered to be attractive sites for insertion of heterologous genes because it is possible to transfer the strongly expressed spheroidin gene, or the thymidine kinase gene, as an expression cassette, not only in insect cells, but for use in vertebrate poxviruses such as vaccinia and swinepox virus.
Another aspect of the subject invention pertains to the use of the entomopoxvirus spheroidin or thymidine kinase gene regulatory sequences in other virus vector systems to enhance the performance of those systems. Thus, the subject invention further pertains to the use of regulatory elements from entomopoxvirus to construct novel chimeric vaccines and expression systems which are functional across genera of mammalian poxviruses.
As one aspect, the invention provides novel Entomopoxvirus polynucleotide sequences, free from other viral sequences with which the Entomopoxvirus sequences are associated in nature. Specifically, the subject invention provides nucleotide sequences of Entomopoxvirus spheroidin and thymidine kinase genes, including flanking sequences and regulatory sequences. In particular embodiments, the spheroidin DNA sequence is that which occurs in the Choristoneura biennis, Choristoneura fumiferana, or Amsacta moorei Entomopoxviruses. Also specifically exemplified is the Amsacta moorei Entomopoxvirus thymidine kinase nucleotide sequence. As explained more fully herein, fragments and variants of the exemplified sequences are within the scope of the subject invention. Fragments and variants can be any sequence having substantial homology with the exemplified sequences so long as the fragment or variant retains the utility of the exemplified sequence. One specific type of variant pertains to spheroidin or tk genes from Entomopoxviruses other than those specifically exemplified herein. As described herein, for example, the current inventors have discovered that the spheroidin genes are highly conserved among different species of Entomopoxvirus. Specifically exemplified herein are three different Entomopoxviruse spheroidin genes having a high degree of homology. Other such spheroidin variants or tk variants from other Entomopoxviruses could be readily located and used by the ordinarily skilled artisan having the benefit of the subject application.
As another aspect, the present invention provides recombinant polynucleotide sequences comprising a sequence encoding an Entomopoxvirus spheroidin protein and/or its regulatory sequences, or a variant or fragment of the spheroidin sequence, linked to a second polynucleotide sequence encoding a heterologous gene. One embodiment of such a polynucleotide sequence provides a spheroidin promoter sequence operably linked to a heterologous gene to direct the expression of the heterologous gene in a selected host cell. Another embodiment provides a sequence encoding a spheroidin protein linked to the heterologous gene in a manner permitting expression of a fusion protein. Still another embodiment provides the heterologous gene inserted into a site in a spheroidin gene so that the heterologous gene is flanked on both termini by spheroidin sequences.
Yet a further aspect of the invention provides a recombinant polynucleotide sequence encoding an Entomopoxvirus tk gene and/or its regulatory sequences, or a variant or fragment thereof, linked to a second polynucleotide sequence encoding a heterologous gene. One embodiment of this polynucleotide sequence provides the tk promoter sequence operably linked to the heterologous gene to direct the expression of the heterologous gene in a selected host cell. Another embodiment provides the sequence encoding the tk protein linked to the heterologous gene in a manner permitting expression of a fusion protein. Still another embodiment provides the heterologous gene inserted into a site in the tk gene so that the heterologous gene is flanked on both termini by tk sequences.
Another aspect of the invention pertains to Entomopoxvirus spheroidin polypeptides, fragments thereof, or analogs thereof, optionally fused to a heterologous protein or peptide. Also provided is an Entomopoxvirus tk polypeptide, fragments thereof, or analogs thereof, optionally linked to a heterologous protein or peptide.
Yet another aspect of the invention is provided by recombinant polynucleotide molecules which comprise one or more of the polynucleotide sequences described above. This molecule may be an expression vector or shuttle vector. The molecule may also contain viral sequences originating from a virus other than the Entomopoxvirus which contributed a spheroidin or tk polynucleotide sequence, e.g., vaccinia.
In another aspect, the present invention provides a recombinant virus comprising a polynucleotide sequence as described above. Also provided are host cells infected with one or more of the described recombinant viruses.
The present invention also provides a method for producing a selected polypeptide comprising culturing a selected host cell infected with a recombinant virus, as described above, and recovering said polypeptide from the culture medium.
In another aspect, the invention provides a method for screening recombinant host cells for insertion of heterologous genes comprising infecting the cells with a recombinant virus containing a polynucleotide molecule comprising the selected heterologous gene sequence linked to an incomplete spheroidin or tk polynucleotide sequence or inserted into and interrupting the coding sequences thereof so that the heterologous gene is flanked at each termini by an Entomopoxvirus spheroidin or tk polynucleotide sequence. The absence of occlusion bodies formed by the expression of a spheroidin protein in the spheroidin-containing cell indicates the integration of the heterologous gene. Alternatively, the absence of the thymidine kinase function, i.e., resistance to methotrexate or a nucleotide analogue of methotrexate, formed by the integration of the inactive thymidine kinase sequence indicates the insertion of the heterologous gene.
In another aspect, the invention provides a shuttle vector system that facilitates expression of heterologous genes in insect or mammalian cells.
Other aspects and advantages of the present invention are described further in the following detailed description of embodiments of the present invention.
SEQ ID NO. 1 is the DNA sequence of the Amsacta moorei Entomopoxvirus spheroidin gene and flanking sequences (shown in FIGS. 2 and 7).
SEQ ID NO. 2 is the amino acid sequence encoded by the G1L ORF (shown right to left in FIG. 2).
SEQ ID NO. 3 is the amino acid sequence encoded by the G2R ORF (shown in FIG. 2).
SEQ ID NO. 4 is the amino acid sequence encoded by the G3L ORF (shown right to left in FIG. 2).
SEQ ID NO. 5 is the amino acid sequence encoded by the G4R ORF (shown in FIG. 2).
SEQ ID NO. 6 is the deduced amino acid sequence of the spheroidin protein (shown as G5R ORF in FIG. 2).
SEQ ID NO. 7 is the amino acid sequence encoded by the partial G6L ORF (shown right to left in FIG. 2).
SEQ ID NO. 8 is the DNA sequence of the Amsacta moorei Entomopoxvirus thymidine kinase (tk) gene (Q2 ORF) and flanking sequences (shown in FIG. 3).
SEQ ID NO. 9 is a small peptide of 66 amino acids potentially encoded by ORF Q1 (shown right to left in FIG. 3).
SEQ ID NO. 10 is the deduced amino acid sequence of the tk protein (from Q2 ORF; shown right to left in FIG. 3).
SEQ ID NO. 11 is the amino acid sequence encoded by Q3 ORF (shown in FIG. 3).
SEQ ID NO. 12 is the synthetic oligonucleotide designated RM58.
SEQ ID NO. 13 is the synthetic oligonucleotide designated RM82.
SEQ ID NO. 14 is the synthetic oligonucleotide designated RM83.
SEQ ID NO. 15 is the synthetic oligonucleotide designated RM92.
SEQ ID NO. 16 is the synthetic oligonucleotide designated RM118.
SEQ ID NO. 17 is the synthetic oligonucleotide designated RM165.
SEQ ID NO. 18 is the synthetic oligonucleotide designated RM03.
SEQ ID NO. 19 is the synthetic oligonucleotide designated RM04.
SEQ ID NO. 20 is the synthetic oligonucleotide designated RM129.
SEQ ID NO. 21 is the spheroidin gene coding sequence (G5L ORF) spanning nucleotides #3080 through #6091 of SEQ ID NO. 1.
SEQ ID NO. 22 is a fragment of the spheroidin gene spanning nucleotides #2781 through 3199 of SEQ ID NO. 1 which is likely to contain the promoter sequence.
SEQ ID NO. 23 is the G2R ORF (shown in FIG. 2).
SEQ ID NO. 24 is the G4R ORF (shown in FIG. 2).
SEQ ID NO. 25 is the G1L ORF (shown in FIG. 2).
SEQ ID NO. 26 is the G3L ORF (shown in FIG. 2).
SEQ ID NO. 27 is the partial G6L ORF (shown in FIG. 2).
SEQ ID NO. 28 is the tk gene coding sequence (Q2 ORF) spanning nucleotides #234 through #782 of SEQ ID NO. 8.
SEQ ID NO. 29 is a fragment flanking the tk gene spanning nucleotides #783 through #851 of SEQ ID NO. 8.
SEQ ID NO. 30 is a fragment spanning nucleotides #750 through #890 of SEQ ID NO. 8 which is likely to contain the promoter sequence.
SEQ ID NO. 31 is the Q1 ORF (shown in FIG. 3).
SEQ ID NO. 32 is the Q3 ORF (shown in FIG. 3).
SEQ ID NO. 33 is a fragment included within the sequence spanning nucleotides #2274 through #6182 of SEQ ID NO. 1 containing the entire spheroidin open reading frame and some flanking sequences.
SEQ ID NO. 34 is a polypeptide cleavage product according to the subject invention.
SEQ ID NO. 35 is a polypeptide cleavage product according to the subject invention.
SEQ ID NO. 36 is a polypeptide cleavage product according to the subject invention.
SEQ ID NO. 37 is the peptide sequence encoded by the RM58 probe.
SEQ ID NO. 38 is a nucleotide fragment spanning nucleotides #4883 through #4957 of SEQ ID NO. 1.
SEQ ID NO. 39 is a nucleotide fragment spanning nucleotides #3962 through #4012 of SEQ ID NO. 1.
SEQ ID NO. 40 is a nucleotide fragment spanning nucleotides #4628 through #4651 of SEQ ID NO. 1.
SEQ ID NO. 41 is the partial AmEPV NPH I nucleotide sequence (shown in FIG. 7).
SEQ ID NO. 42 is the partial AmEPV NPH I amino acid sequence (shown right to left in FIG. 7).
SEQ ID NO. 43 is the CbEPV nucleotide sequence shown in part A of FIG. 6.
SEQ ID NO. 44 is the CbEPV amino acid sequence shown in part B of FIG. 6.
SEQ ID NO. 45 is the CfEPV nucleotide sequence shown in part A of FIG. 6.
SEQ ID NO. 46 is the CfEPV amino acid sequence shown in part B of FIG. 6.
SEQ ID NO. 47 is the CbEPV amino acid sequence corresponding to amino acids 211 to 221 of AmEPV (shown in part C of FIG. 6).
SEQ ID NO. 48 is the CbEPV amino acid sequence corresponding to amino acids 682 to 691 of AmEPV (shown in part C of FIG. 6).
SEQ ID NO. 49 is the CbEPV amino acid sequence corresponding to amino acids 726-736 of AmEPV (shown in part C of FIG. 6).
SEQ ID NO. 50 is the synthetic oligonucleotide designated RM206.
SEQ ID NO. 51 is the synthetic oligonucleotide designated RM212.
SEQ ID NO. 52 is the synthetic oligonucleotide designated RM58.
SEQ ID NO. 53 is the synthetic oligonucleotide designated RM75.
SEQ ID NO. 54 is the synthetic oligonucleotide designated RM76.
SEQ ID NO. 55 is the synthetic oligonucleotide designated RM78.
SEQ ID NO. 56 is the synthetic oligonucleotide designated RM79.
SEQ ID NO. 57 is the synthetic oligonucleotide designated RM87.
SEQ ID NO. 58 is the synthetic oligonucleotide designated RM91.
SEQ ID NO. 59 is the synthetic oligonucleotide designated RM93.
SEQ ID NO. 60 is the synthetic oligonucleotide designated RM95.
SEQ ID NO. 61 is the synthetic oligonucleotide designated RM169.
SEQ ID NO. 62 is the synthetic oligonucleotide designated RM170.
SEQ ID NO. 63 is the synthetic oligonucleotide designated RM282.
SEQ ID NO. 64 is the synthetic oligonucleotide designated RM283.
SEQ ID NO. 65 is the synthetic oligonucleotide designated pTk1.
SEQ ID NO. 66 is the synthetic oligonucleotide designated pTk2.
SEQ ID NO. 67 is the synthetic oligonucleotide designated pTk3.
SEQ ID NO. 68 is the synthetic oligonucleotide designated pTk4.
SEQ ID NO. 69 is the synthetic oligonucleotide designated pU.
SEQ ID NO. 70 is the synthetic oligonucleotide designated pU2.
SEQ ID NO. 71 is the synthetic oligonucleotide designated pU20.
SEQ ID NO. 72 is the synthetic oligonucleotide designated pD 1.
SEQ ID NO. 73 is the synthetic oligonucleotide designated pD2.
SEQ ID NO. 74 is the complete AmEPV NPH I (G6L ORF) nucleotide sequence (shown in FIGS. 2 and 7).
SEQ ID NO. 75 is the complete AmEPV NPH I amino acid sequence (shown right to left in FIGS. 2 and 7).
SEQ ID NO. 76 is the AmEPV nucleotide sequence (shown in part A of FIG. 6).
SEQ ID NO. 77 is the AmEPV amino acid sequence (shown in part B of FIG. 6).
SEQ ID NO. 78 is the RM58 binding site found in the spheroidin gene.